Crystallization and preliminary X-ray diffraction data for a purple acid phosphatase from sweet potato

Purple acid phosphatase from sweet potato is a homodimer of 110 kDa. Two forms of the enzyme have been characterized. One contains an Fe±Zn centre similar to that previously reported for red kidney bean purple acid phosphatase. Another isoform, the subject of this work, is the ®rst con®rmed example of an Fe±Mn-containing enzyme. Crystals of this protein have been grown from PEG 6000. They have unit-cell parameters a = b = 118.4, c = 287.4 A Ê and have the symmetry of space group P6522, with one dimer per asymmetric unit. Diffraction data collected using a conventional X-ray source from a cryocooled crystal extend to 2.90 A Ê resolution. The three-dimensional structure of the enzyme will provide insight into the coordination of this novel binuclear metal centre

Inhibition of purple acid phosphatase with a-alkoxynaphthylmethylphosphonic acids

Purple acid phosphatases (PAPs) are binuclear hydrolases that catalyse the hydrolysis of a range of phosphorylated substrates. Human PAP is a major histochemical marker for the diagnosis of osteoporosis. In patients suffering from this disorder, PAP activity contributes to increased bone resorption and, therefore, human PAP is a key target for the development of anti-osteoporotic drugs. This manuscript describes the design and synthesis of derivatives of 1-naphthylmethylphosphonic acids as inhibitors of PAP. The Ki values of these compounds are as low as 4 lM, the lowest reported to date for a PAP inhibitor

Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

BACKGROUND Tartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics. FINDINGS A new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine. CONCLUSION The gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism.This work was funded by the Royal Society of Tropical Medicine and Hygiene through a Dennis Burkitt Fellowship to JJM. ARD is supported by the Economic and Social Research Council. JJM is supported by a Wellcome Trust Research Training Fellowship (GR074833MA)

The organophosphate-degrading enzyme from Agrobacterium radiobacter displays mechanistic flexibility for catalysis

The OP (organophosphate)-degrading enzyme from Agrobacterium radiobacter (OpdA) is a binuclear metallohydrolase able to degrade highly toxic OP pesticides and nerve agents into less or non-toxic compounds. In the present study, the effect of metal ion substitutions and site-directed mutations on the catalytic properties of OpdA are investigated. The study shows the importance of both the metal ion composition and a hydrogenbond network that connects the metal ion centre with the substrate-binding pocket using residues Arg254 and Tyr257 in the mechanism and substrate specificity of this enzyme. For theCo(II) derivative of OpdA two protonation equilibria (pKa1 ∼5; pKa2 ∼10) have been identified as relevant for catalysis, and a terminal hydroxide acts as the likely hydrolysis-initiating nucleophile. In contrast, the Zn(II) and Cd(II) derivatives only have one relevant protonation equilibrium (pKa ∼4–5), and theμOHis the proposed nucleophile. The observed mechanistic flexibility may reconcile contrasting reaction models that have been published previously and may be beneficial for the rapid adaptation of OP-degrading enzymes to changing environmental pressures

Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle

Background: Purple acid phosphatases belong to the family of binuclear metallohydrolases and are involved in a multitude of biological functions, ranging from bacterial killing and bone metabolism in animals to phosphate uptake in plants. Due to its role in bone resorption purple acid phosphatase has evolved into a promising target for the development of anti-osteoporotic chemotherapeutics. The design of specific and potent inhibitors for this enzyme is aided by detailed knowledge of its reaction mechanism. However, despite considerable effort in the last 10 years various aspects of the basic molecular mechanism of action are still not fully understood

Crystal structure of plant acetohydroxyacid synthase, the target for several commercial herbicides

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the first enzyme in the branched-chain amino acid biosynthesis pathway. Five of the most widely used commercial herbicides (i.e. sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl-benzoates and sulfonylamino-cabonyl-triazolinones) target this enzyme. Here we have determined the first crystal structure of a plant AHAS in the absence of any inhibitor (2.9 Å resolution) and it shows that the herbicide-binding site adopts a folded state even in the absence of an inhibitor. This is unexpected because the equivalent regions for herbicide binding in uninhibited Saccharomyces cerevisiae AHAS crystal structures are either disordered, or adopt a different fold when the herbicide is not present. In addition, the structure provides an explanation as to why some herbicides are more potent inhibitors of Arabidopsis thaliana AHAS compared to AHASs from other species (e.g. S. cerevisiae). The elucidation of the native structure of plant AHAS provides a new platform for future rational structure-based herbicide design efforts. Database: The coordinates and structure factors for uninhibited AtAHAS have been deposited in the Protein Data Bank (www.pdb.org) with the PDB ID code 5K6Q

Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase

ABSTRACT: Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 Å), sulfometuron methyl (2.34 Å), and two other sulfonylureas, metsulfuron methyl (2.29 Å) and tribenuron methyl (2.58 Å). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethylThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme

Purple acid phosphatase inhibitors as leads for osteoporosis chemotherapeutics

Purple acid phosphatases (PAPs) are metalloenzymes that catalyse the hydrolysis of phosphate esters under acidic conditions. Their active site contains a Fe(III)Fe(II) metal centre in mammals and a Fe(III)Zn(II) or Fe(III)Mn(II) metal centre in plants. In humans, elevated PAP levels in serum strongly correlate with the progression of osteoporosis and metabolic bone malignancies, which make PAP a target suitable for the development of chemotherapeutics to combat bone ailments. Due to difficulties in obtaining the human enzyme, the corresponding enzymes from red kidney bean and pig have been used previously to develop specific PAP inhibitors. Here, existing lead compounds were further elaborated to create a series of inhibitors with K values as low as ∼30 μM. The inhibition constants of these compounds were of comparable magnitude for pig and red kidney bean PAPs, indicating that relevant binding interactions are conserved. The crystal structure of red kidney bean PAP in complex with the most potent inhibitor in this series, compound 4f, was solved to 2.40 Å resolution. This inhibitor coordinates directly to the binuclear metal centre in the active site as expected based on its competitive mode of inhibition. Docking simulations predict that this compound binds to human PAP in a similar mode. This study presents the first example of a PAP structure in complex with an inhibitor that is of relevance to the development of anti-osteoporotic chemotherapeutics

Deacidification of grass silage press juice by continuous production of acetoin from its lactate via an immobilized enzymatic reaction cascade

An immobilized enzymatic reaction cascade was designed and optimized for the deacidification of grass silage press juice (SPJ), thus facilitating the production of bio-based chemicals. The cascade involves a three-step process using four enzymes immobilized in a Ca-alginate gel and uses lactic acid to form acetoin, a value-added product. The reaction is performed with a continuous, pH-dependent substrate feed under oxygenation. With titrated lactic acid yields of up to 91% and reaction times of ca. 6 h was achieved. Using SPJ as titrant yields of 49% were obtained within 6 h. In this deacidification process, with acetoin one value-added bio-based chemical is produced while simultaneously the remaining press juice can be used in applications that require a higher pH. Such, this system can be applied in a multi-product biorefinery concept to take full advantage of nutrient-rich SPJ, which is a widely available and easily storable renewable resource